1. Cell Signaling
Cells use a large number of clearly defined signaling pathways to regulate their activity. These signaling pathways fall into two main groups depending on how they are activated. Most of them are activated by external stimuli and function to transfer information from the cell surface to internal effector systems. However, some of the signaling systems respond to information generated from within the cell, usually in the form of metabolic messengers. For all of these signaling pathways, information is conveyed either by protein-protein interactions or it is transmitted by diffusible elements usually referred to as second messengers or transmitters. The ability of cells to perceive and correctly respond to other cells is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes.
2. Wnt Proteins and Frizzled Receptors
Secreted Wnt (Wg Int) proteins have numerous signaling functions during development, mediated by certain receptors on the cell surface. These proteins are defined by their sequence rather than by functional properties. They contain a signal sequence of 350-380 amino acids followed by a highly conserved distribution of cysteines. Although Wnt proteins are secreted they show an insoluble nature that has been explained by the discovery that these proteins are palmitoylated and are more hydrophobic than initially predicted from the primary amino acid sequence. The palmitoylation is found on a conserved cysteine, suggesting that all Wnts essentially carry this modification (Willert K. et al., 2003). Until now this insoluble nature of all members of the Wnt family has hindered attempts to purify the Wnts and precluded an isolation of Wnts in high quantities.
Wnts proteins play diverse and essential roles in generation of cell polarity, embryonic induction, specification of cell fate, and diseases such as cancer or degeneration. At the molecular level, Wnt proteins operate largely via receptor-mediated signaling pathways, and these receptors appear to be members of the frizzled family (Huang H. and Klein P., 2004).
Frizzled receptors (Fz) are integral membrane proteins with transmembrane domains, an exposed binding site outside the cell and an effector site extending into the cytosol. They function in multiple signal transduction pathways and have been identified in numerous animals, from sponges to humans. The family is defined by conserved structural features, including seven hydrophobic transmembrane domains and a cysteine rich ligand-binding domain (Huang H. and Klein P., 2004).
Fz function in three distinct signaling pathways, known as the planar cell polarity pathway, the canonical Wnt/β-catenin pathway, and the Wnt/calcium pathway. The cytoplasmic Fz domains that link to heterotrimeric G proteins and other downstream signaling components transduce a signal to several intracellular proteins that include dishevelled, glycogen synthase kinase-3β (GSK-3β), axin, adenomatous polyposis coli (APC), and the transcriptional regulator β-catenin. Cytoplasmic β-catenin levels are normally kept low by continuous proteasome-mediated degradation, which is controlled by a complex containing GSK-3/APC/Axin. When cells receive Wnt signals, the degradation pathway is inhibited, and consequently β-catenin accumulates in the cytoplasm and nucleus. Nuclear β-catenin interacts with transcription factors such as lymphoid enhancer-binding factor 1/T cell-specific transcription factor to affect transcription. A large number of Wnt targets have been identified that include members of the Wnt signal transduction pathway itself, which provide feedback control during Wnt signaling (Logan C. and Nusse R., 2004).
Secreted proteins of the Wnt family play widespread roles in the regulation of embryonic development, and aberrant activation of the canonical Wnt/β-catenin pathway is one of the most frequent signaling abnormalities known in human cancer. In human breast cancer, evidence of β-catenin accumulation implies that the canonical Wnt signaling pathway is active in over 50% of carcinomas (Brennan K. and Brown M., 2004).
Much of last years' research focused on the development of cell based assays and screening techniques for drug discovery and design. Screening of cell signaling pathways in primary cells of a physiologically relevant phenotype provides a means to identify modulators of important disease pathways that lack known drug targets.
Borchert K. et al. (2006) screened for small molecule activators of Fz in primary human preosteoblasts that should contain intact and physiologically relevant Wnt/FZ signaling components. For this purpose they measured endogenous translocation of the downstream transcription factor β-catenin to the nucleus by combining standard immunofluorescent techniques with automated fluorescence microscopy.
DasGupta R. et al. (2005) used the availability of the Drosophila genome sequence, to find new components in the Wnt signaling pathway. By RNA interference (RNAi) based screening technology they identified functional genes regulating the Wnt-Wg pathway. The assay for the RNAi screen was based on the Wnt reporter TOP-Flash (TCF optimal promoter), which consists of multimerized TCF-binding sites driving the expression of a cDNA encoding the firefly luciferase gene. The screen was performed in Drosophila imaginal disc-derived clone 8 cells, which are epithelial in origin. The activity of the Wg signaling pathway was quantified by measurement of normalized luciferase expression or relative luciferase activity units, which equated to the ratio of the absolute activity of firefly luciferase to that of renilla luciferase.
Signal transduction has been studied extensively with cell based systems, but interest and commercial investment in Fz in areas such as drug targets, orphan receptors, high throughput screening, and biosensors, among others will focus greater attention on cell free assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell based assays are adequate for many Fz, these formats would limit the development of higher density Fz assay platforms mandatory for other applications. Stable, robust and cell free signaling assemblies comprising receptor and appropriate molecular switching components form the basis of future Fz assay platforms adaptable for such applications as microarrays. In addition cell free assays ensure a uniform response resulting in well-defined mechanisms of action and no ambiguity of experimental data due to other interfering and interactive pathways within the cell. Another advantage of cell free assays is the free accessibility of the compound to the target.
Thus there is a need for a fast and convenient cell free assay to measure the activity of Fz that is suitable for high throughput screening.
The solution to this problem is achieved by providing the embodiments characterized by the claims, and described further below.